Herbicide-tolerant plants expressing carbamate hydrolase

ABSTRACT

A process for the isolation and characterization of a gene enzyme system for the inactivation of the herbicide phenmedipham, wherein the enzyme is a carbamate hydrolase of Arthrobacter oxidans, which is responsible for the cleavage of the carbamate bond between the benzene rings of phenmedipham. This process includes the isolation of the carbamate hydrolase, the identification of the amino acid sequence of two BrCN cleavage peptides of the carbamate hydrolase, the synthesis of oligonucleotides for specific determination of the carbamate hydrolase sequence by hybridization and identification of the coding region, cloning and specifying the nucleotide sequence of the carbamate hydrolase gene from Arthrobacter oxidans. 
     Plants are transformed with recombinant genes coding for the carbamate hydrolase and transgenic plants which are tolerant to the herbicide are produced.

This application is a continuation-in-part of U.S Ser. No. 07/353,871,filed on May 18, 1989 abandoned.

The present invention relates to a process for the isolation andcharacterisation of a gene enzyme system for the inactivation of theherbicide phenmedipham and transfer of the gene into plants to produceherbicide-tolerant plants. The enzyme is a carbamate hydrolase ofArthrobacter oxidans, which is responsible for the cleavage of thecarbamate bond between the benzene rings of phenmedipham which is thecommon name for the herbicide methyl3-m-tolylcarbamoyloxyphenyl-carbamate.

In practice it is often necessary to use several herbicides or herbicidemixtures to combat various weeds. These problems can be avoided by abiotechnical change to the plant in which resistance to a non-selectiveherbicide is introduced.

The production of herbicide-tolerant plants is now coming more to theforeground in the plant protection area.

In order to produce herbicide-tolerant plants, it is necessary first tohave a process for the isolation and subsequent purification of anenzyme which is able to inactivate a herbicide, e.g. by metabolism, andthen to have a process for characterising the gene enzyme systemcontaining the DNA sequence that codes for the active enzyme. Afterthese steps the DNA sequence of the gene can be transferred into plants.

Such a process for the isolation and subsequent characterisation of agene enzyme system which can inactivate phenmedipham and the transfer ofthis gene enzyme system into plants was not previously known.

It has now been found that a carbamate hydrolase can be isolated fromsome microorganisms, such as Arthrobacter oxidans, which is responsiblefor the hydrolysis of the carbamate bond between the two benzene ringsof phenmedipham. The hydrolysis of this bond leads to herbicidallyinactive compounds such as methyl 3-hydroxyphenylcarbamate andmeta-toluidine, according to the following reaction: ##STR1## Forisolation and subsequent purification of a gene enzyme system which canhydrolyse phenmedipham according to the above described reaction,microorganisms of Arthrobacter oxidans are cultivated in a nutrientmedium. The carbamate hydrolase responsible for the cleavage ofphenmedipham is isolated by ultrasound cell destruction, centrifugationand purification by anion exchange chromatography, gradient elution,ammonium sulphate precipitation and FPLC separation untilelectrophoretic homogeneity is achieved. From the purified carbamatehydrolase, two peptides are isolated after BrCN cleavage whose sequencecan be estimated by Edman degradation. According to the sequenceinformation of these peptides, oligonucleotides can be synthesised whichcan be used as hybridization probes for the detection of the carbamatehydrolase gene.

In all of the mentioned isolates of the soil bacteria Arthrobacteroxidans, plasmids could be detected after lysis of the cells andextraction of the nucleic acids.

For the species Arthrobacter oxidans P52, it can be shown that thecarbamate hydrolase is coded by one plasmid.

With the loss of the plasmid pHP52 the properties of this species to beable to hydrolytically cleave phenmedipham is lost. A Carbamatehydrolosis activity cannot be biochemically established in theplasmid-free derivative of the species P52.

The plasmid pHP52 can be preparatively isolated and mapped withrestriction endonucleases. The electrophoretically separated restrictionfragments can be transferred onto membrane filters and hybridised withvarious oligonucleotides. From the data from the blot-hybridisations, itappears that the carbamate hydrolase gene is localised on a PstIrestriction fragment with a size of 3.3 kb.

This fragment can be preparatively isolated from the plasmid pHP52 andinserted in the PstI position of the vector pUC19C (Yanish-Perron. C.,Vieira, J. & Messing (1985) Gene 33, 103 ff). After transformation of Ecoli DH5α with the ligation preparation, two types of recombinant E.coli clones are obtained (pp52Pst and pp52Pst inv.) which contain thecarbamate hydrolase gene in different orientations to the Lac-promoterof the vector (see FIG. 5).

The carbamate hydrolase can be functionally expressed in the presence ofthe inducer isopropyl-β-D-thiogalactopyranoside from cultures of theclone of the type E. coli DH5α (pp52Pst). In protein extracts of clonesof the type E. coli DH5α (pp52 inv.) which contain the carbamatehydrolase gene in inverse orientation to the Lac-promoter, no expressionof the carbamate hydrolase gene can be detected.

The nuceleotide sequence of the carbamate hydrolase gene can bedetermined according to the method of Sanger et al. (Sanger, F.,Nicklen, S. & Coulson, A (1977), Proc. Natl. Acad. Sci. USA 74,5463-5468).

15 sub-clones arising from the cloning of 3.3 kb long PstI restrictionfragments can be constructed in the single strain DNA bacteriophages M13mp 18 and mp 19 (Messing, J. (1983) Methods in Enzymol. 101, 20-78). InFIG. 6 an exact restriction map of the coded area is represented fromwhich the sequencing strategy can be seen.

The established nucleotide sequence (SEQ ID NO:7) with the proteinsequence thus derived is illustrated in FIG. 7 (SEQ ID NO:6). The aminoacid sequences of both established BrCN-splitting peptides (see Example4) can be identified in the same reading frame as the DNA level. Thisreading frame ends with a TGA translation stop codon (see FIG. 7 (SEQ IDNO:6), nucleotide positions 1789-1791) and begins very probably with aGTG-start codon (FIG. 7 (SEQ ID NO:6) - nucleotide positions 310-312).Altogether a reading frame of 1479 base pairs results. Upstream of theputative GTG-start codon, a region with significant homology to theconsensus sequence for E. coli ribosome binding sites ("Shine-DalgarnoBox") can be established (see FIG. 7 (SEQ ID NO:6), nucleotide positions298-302).

After the nucleotide sequence of the carbamate hydrolase gene has beendetermined, the construction of plasmids for the expression of carbamatehydrolase in plants can be carried out (see Example 9). For this thechimeric carbamate hydrolase gene on plasmids can be transfered from E.coli to Aqrobacterium tumefaciens and from Aqrobacterium tumefaciens tothe target plants (see Example 10). The operation of the carbamatehydrolase gene in transformed plants can be shown by sprayingtransformed and untransformed plants with phenmedipham (see Examples 11and 12).

On the 25th August 1987 the following micro-organisms were deposited atthe German Collection of Microorganisms (DSM) in Gottingen, Germany.

    ______________________________________                                        Arthrobacter oxidans                                                                       P 16/4/B       (DSM 4038)                                        Arthrobacter oxidans                                                                       P 67           (DSM 4039)                                        Arthrobacter oxidans                                                                       P 75           (DSM 4040)                                        Arthrobacter oxidans                                                                       P 11/1/-b      (DSM 4041)                                        Arthrobacter oxidans                                                                       P 15/4/A       (DSM 4045)                                        Arthrobacter oxidans                                                                       P 21/2         (DSM 4046)                                        Arthrobacter oxidans                                                                       P 52, containing                                                                             (DSM 4044).                                                    the plasmid pHP52                                                ______________________________________                                    

Abbreviations

DEAE Diethylaminoethyl

FPLC East protein/peptide/polynucleotide liquid Chromatography

SDS Sodium lauryl sulphate

DTT Dithiothreitol

1×SSC 0.15M NaCl 0.015M trisodium citrate pH 7.0

1×Denhardt 0.02% (w/v) Bovine serum albumin (Sigma. Fraction V) 0.02%(w/v) Ficoll 400 0.02% polyvinylpyrrolidone

MCS Multiple cloning site

Abbreviation for restriction endonucleases

Bm=BamHI, Bs=BstEII. Cl=ClaI, EV=EcoRV, HII=HindII, Kp=KpnI, Nc=NcoI,Nd=NdeI, Nh=NheI, Ps=PstI, PvI=PvuI, PvII=PvuII, Sc=SacI, Sp=SphI,St=StuI. Xb=XbaI.

DESCRIPTION OF THE FIGURE

Figures

FIG. 1 shows the process for isolating and subsequent purification ofphenmedipham cleaving carbamate hydrolase from Arthrobacter oxidans(Example 2)

FIG. 2 shows the electrophoretic separation of the crude extract and theisolation and pooled protein fractions after the individual purificationsteps on an SDS-polyacrylamide gel, in which standard proteins (M) runalongside as markers for the molecular weight.

A: Crude extract from the centrifuge supernatant of the ultrasound celldestruction of Arthrobacter oxidans.

B: Pooled protein fraction after gradient elution on a DEAE Sephacelcolumn.

C: Ammonium sulphate precipitation of the fraction from B.

D: Protein fraction after gel filtration of the ammonium sulphateprecipitation and subsequent separation on a Sephacryl S-300 column.

E: Protein fraction after separation of the pooled fraction from D byFPLC (anion exchange chromatography).

F: Protein fraction after separation of the pooled fraction E on aSuperose 6 column (Example 2).

FIG. 3 shows a diagram from which the pH-optimum (pH 6.8) of thecarbamate hydrolase can be obtained. The pH optimum can be establishedin such a way that the enzyme activity as a percentage against the pHvalue can be derived (Example 2).

FIG. 4 shows the restriction map of the plasmid pHP52 from theArthrobacter oxidans (species P52).

FIG. 5 shows a scheme of cloning of the carbamate hydrolase gene. Toimprove clarity the regions of the gene which hybridize witholigonucleotides used including the 5' and 3' flanking regions areenlarged above the ring-forming plasmid map.

The recombinant clones pp52Pst and pp52Pst inv. are represented in alinear manner. The transcription direction of the lac Z gene isrepresented by an arrow. ( ).

FIG. 6 shows the restriction map of the cloned 3.3 kb PstI restrictionfragment which arises from the exact position of the carbamate hydrolasegene (Start: GTG=Start codon. Stop: TGA=Stop codon).

The sequenced areas are characterised by arrows under the restrictionmap. From the length of the arrow can be read the length of theindividual sequenced areas.

The restriction fragments which contain the homology areas of theoligonucleotides described in Example 4 are emphasised in the map. ( )

The M13 clones are described as follows:

    ______________________________________                                        Clone          Inserted Fragment M13 Vector                                   ______________________________________                                        A     NdeI/SacI (pUC19)                                                                              ˜500  mp 18                                      B     NdeI/SacI        1289    bp  mp 18                                      C     PvuI/PstI        ˜700                                                                            bp  mp 19                                      D     PvuI/PvuI        564     bp  mp 18                                      E     BamHI/BamHI (pUC19)                                                                            ˜1060                                                                           bp  mp 18                                      F     BamHI/BamHI      958     bp  mp 18                                      G     PvuI/PvuI        564     bp  mp 18                                      H     PvuII/SacI       476     bp  mp 18                                      I     ClaI/BamHI       561     bp  mp 18                                      K     ClaI/BamHI       397     bp  mp 18                                      L     PvuII/SacI       476     bp  mp 19                                      M     KpnI/HindII      445     bp  mp 19                                      N     BamHI/BamHI      958     bp  mp 18                                      O     BamHI/BamHI      1100    bp  mp 18                                      P     KpnI/HindII      445     bp  mp 18                                      ______________________________________                                    

FIG. 7 (SEQ ID NO:6) shows the nucleotide sequence of the carbamatehydrolase gene including the 5' and 3' flanking area.

Particularly characterised are:

1. The GTG start codon

2. The ribosomal binding site (Shine-/Dalgarno Box: S/D)

3. The homology area of the oligonucleotide described in Example 4(oligo I and II/oligo III).

The coding nucleotide sequence was formally translated as an amino acidsequence. The reading frame is determined clearly by the protein levelof the established amino acid partial sequences,

FIG. 8 shows the construction of plasmid pUPA1014. Starting from a 3.3kb PstI fragment that originates from plasmid pHP52 (example 7) andcontains the complete bacterial gene for phenmedipham carbamatehydrolase (PMPH), the untranslated 3'-region of the fragment is replacedby 0.2 kb fragment from the Aqrobacterium tumefaciens T-DNA containingthe octopine synthase gene (OCS) polyadenylation signal (pA). Thevectors used are pUC19(for pp52Pst) and pUC18 (for pA5 and pUPA1014).

FIG. 9 shows the construction of plasmid pUP01015. In this cloning stepthe untranslated 5'-region and the N-terminal part of the coding regionis removed from the carbamate hydrolase gene and replaced by a syntheticdouble-stranded oligonucleotide, which reconstitutes the sequence codingfor the N-terminal part of the protein. The sequence surrounding thetranslational start codon is optimal for plant gene expression accordingto Lutcke et al. EMBO J. 6:43-48, 1987.

FIG. 10 shows the construction of plasmid pMCP01021. In this cloningstep, the coding sequence of the recombinant carbamate hydrolase gene islinked at its 5'-end to the cauliflower mosaic virus (CaMV) 35S promotercontained in plasmid pA8.

FIG. 11 shows the construction of plasmid pMA1017. In this cloning stepa fragment containing the sequence that codes for the potato proteinaseinhibitor II signal peptide is inserted in correct orientation betweenthe CaMV 35S promotor and the OCS polyadenylation signal of plasmid pA8to create an expression vector for targeting introduced genes into theendoplasmatic reticulum of plant cells.

FIG. 12 shows the construction of plasmid pMAP01022. In this cloningstep the coding sequence of the recombinant carbamate hydrolase gene islinked at its 5'-end in the correct reading frame to the signal peptidesequence contained in plasmid PMA1017.

FIG. 13 shows transgenic tobacco plants WS28-19 in comparison withuntransformed control tobacco W38 three weeks after treatment with theherbicide phenmedipham. Photographs A, B, C, show control plants (left)which were sprayed with 1 kg/ha, transgenic plants (middle) which weresprayed with 1 kg/ha (A), 3 kg/ha (B) and 10 kg/ha (C) or transgenicplants (right) which were not treated.

FIG. 14 shows transgenic tobacco plants WS28-19 in comparison withuntransformed control tobacco W38 three weeks after treatment with theherbicide phenmedipham. Photograph D shows untransformed control plants(left) and transgenic plants (right) which were treated with 1 kg/haphenmedipham (upper row), or left untreated (lower row), respectively.

FIG. 15 shows the relative variable fluorescence of intact leaves from acontrol W38 plant and a transgenic plant WS28-19 after treatment with 1kg/ha phenmedipham. Measurements were made at indicated intervals afterspraying (t=0) as described in the text. Between measurements plantswere further incubated in the growth chamber under 16 hours light and 8hours dark.

EXAMPLE 1

Isolation of microorganisms that possess the ability to inactivate theherbicide phenmedipham.

To identify microorganisms which possessed the ability to inactivate theherbicide phenmedipham by metabolisn, various microorganisms werescreened. As source for the microorganisms, soil samples from variouslocations (field test sites which had been treated several times withphenmedipham), and also from settling sediment, were used. Selectioncriteria for the identification of microorganisms which can carry out acarbamate cleavage, were as follows.

a) Growth in a nutrient medium with phenmedipham as a single carbon ornitrogen source.

b) Breaking down of phenmedipham to highly water soluble compoundsaccording to the following reaction. ##STR2##

From the large number of microorganisms obtained from the soil samples,which were capable of cleavage of phenmedipham, seven representativeswere chosen which clearly showed a breakdown. These soil bacteria whichare all representatives of the Arthrobacter species and within thisspecies, the sub-species of oxidans, were cultivated in culture brothscontaining a synthetic medium (M9-medium) having the followingcomposition:

1.0 g/l NH₄ Cl

0.25 g/l MgSO₄.7H₂ O

3.0 g/l KH₂ PO₄

7.0 g/l Na₂ HPO₄.2H₂ O

2.0 g/l Glucose

and

0.5 g/l NaCl.

The M9-medium, in addition, contained 1 mg/l thiamine (vitamin B1) aswell as trace elements which were added in the form of a stock solution(1 ml/l Mg-medium). The trace element stock solution contained:

0.5 Boric acid

0.04 g/l CuSO₄.5H₂ O

0.2 g/l FeCl₃.6H₂ O

0.4 g/l MnSO₄.7H₂ O

0.4 g/l ZnCl₂

0.2 g/l (NH₄)₆ Mo₇ O₂₄.4H₂ O

For shaking cultures in liquid mediums, the synthetic medium wassupplemented by 0.1% casamino acids (Difco®).

The soil bacteria were incubated in this M9 medium at 28° C. with goodaeration until the end of the logarithmic growth phase. For enzymepurification, a total of 10 liters of medium were inoculated with astationary pre-culture (1:100).

By HPLC analysis of the culture broth it was shown that in the culturesof Arthrobacter oxidans, the desired cleavage of phenmedipham to theherbicidally inactive products was achieved.

EXAMPLE 2

Isolation and purification of the carbamate hydrolase from Arthrobacteroxidans.

The isolation and subsequent purification of the carbamate hydrolase toelectrophoretic homogeneity was carried out over a six stagepurification process. From 6 liters of an end logarithmic culture ofArthrobacter oxidans (pHP52) (DSM No 4044), 0.5-1 mg carbamate hydrolasewas reproducibly isolated. For isolation of the carbamate hydrolase, thecells were harvested by centrifugation (7000×g) and resuspended in about40 ml of decomposition buffer (10 mM sodium phosphate pH 6.8/1 mM DTT).The cell suspension was disrupted by ultrasound and homogenised at thesame time. The homogenate was then centrifuged for 45 minutes at40000×g, at 4° C. The sediment was removed and the supernatant,equilibrated with 100 mM Tris-HCl pH 7.2/100 mM NaCl/1 mM DTT, wasapplied to a DEAE Sephacel column (column diameter 2.6 mm, height of thegel bed 20.5 cm, column volume about 100 ml). Before application to thecolumn, the cell extract was diluted at a ratio of about 1:10 withstarting buffer (100 mM Tris/100 mM NaCl/1 mM DTT). The column was thenwashed with starting buffer in order to remove the unbound material. Thecarbamate hydrolase was then eluted with a linear gradient 100 mM NaCl-500 mM NaCl (5×column volume). The enzymatically active fractions werepooled and treated with dry ammonium sulphate (NH₄)₂ SO₄ to an endconcentration of 33% of the saturated solution. The resulting proteinprecipitate was sedimented by centrifugation (20000×g/30 mins) anddiscarded. The supernatant was treated with solid ammonium sulphate toan end concentration of 60% of the saturated solution and stirred forabout 12 hours at O° C. The sedimented protein was collected bycentrifugation (20000×g/30 mins) and dissolved in about 1 ml startingbuffer, treated with 10% (w/v) saccharose and loaded to a SephacrylS-300 column. The gel filtration was carried out at a flow rate of 2.5cm/h (elution buffer -: start buffer). The column had a diameter of 2.6cm, a height of 95 cm and a volume of 475 ml. The enzymatically activefraction was then worked up on an FPLC column (mono Q HR 5/5; anionexchange). Gradient elution 100 mM NaCl -: 300 mM NaCl: flow rate: 0.5ml/mm; application volume 2 ml). The unbound protein was separated byisocratic elution with 19 ml starting buffer. (Gradient elution 100 mMNaCl -: 300 mM NaCl in 20 ml with 100 mM Tris/HCl pH 7.2/l mM DTT).

The enzymatically active fractions were concentrated by ultra-filtrationafter electrophoretic analysis of the purity (SDS-polyacrylamide-gelelectrophoresis by the method of Lammli), using an Amicon®, Centrikon 10concentrator, and put on a FPLC gel-filtration column (Superose 6-HR10/30, Pharmacia) (flow rate 0.2 ml/min; application volume 100 ul;eluent: 100 mM Tris/HCl pH 7.2/10 mM NaCl).

The active protein fractions which result from this step areelectrophoretically homogenous.

The isolated enzyme is active in buffered solutions (i.e. buffersconventionally used in biochemical systems, such as phosphate buffers.Tris buffers etc; pH 6.8). Co-factors or metal ions are not necessaryfor the reaction. A sensitivity against SH reagents is also not seen.The optimum pH of the enzyme is 6.8.

The molecular weight of the carbamate hydrolase is in the range of50-60. preferably 53-57 kd, both under denaturing/dissociatingconditions (SDS gel electrophoresis) as well as under native conditions(gel-filtration) From this it follows that the carbamate hydrolase is amonomeric protein. The isoelectric point of the carbamate hydrolase isat pI=6.2.

EXAMPLE 3

Process for detecting the carbamate hydrolase.

For a quick and sure determination of enzyme activity during thepurification of the crude protein extracts, an in vitro enzyme test wasdeveloped. The test is based on the ability of the enzyme to change thehighly water insoluble phenmedipham into a soluble hydrolysis product.For this, solid phenmedipham was suspended in water and micronised byultrasound. This micro-suspension was then poured, with stirring at 50°C., into an agarose solution and this mixture put into a petri dishbefore it solidified, where it formed into a turbid gel matrix. Theenzyme solution was then put into wells which had been punched in thesolid matrix. After incubation of the test plates for 2-4 hours at 30°C., the enzyme activity was demonstrated by observing clear zones in thematrix which had been made opaque by the phenmedipham.

EXAMPLE 4

Identification of the amino acid sequence of two BrCN cleaving peptidesand synthesis of oligonucleotides for specific evidence of the carbamatehydrolase gene by hybridization.

Resulting from the purified carbamate hydrolase, two peptides wereisolated after BrCN cleavage, whose partial sequence was established byEdman degradation.

BrCN Peptide I (SEQ ID NO:1):

H₂ N - Ser - Asp - Glu - Phe - Ala - Asn -Leu - Asp - Arg - Trp - Thr -Gly - Lys - Pro - Phe - Val - Asp (Val) - Gly (His) -Leu - Asp - Glu -Val - Ale - Val - COOH

BrCN Peptide II (SEQ ID NO:2):

N₂ H - Glu - His - Thr - Lys - Phe(Val) - Asn(Gly) - Glu - Arg(Cys) -Pro - Leu - Ala - Phe - Tyr - Pro -Val - Phe - Asn - Glu - COOH

According to the amino acid sequence information of these peptides,oligonucleotides were synthesised which could be used as hybridizationprobes for the detection of the carbamate hydrolase gene:

Oligonucleotide I (SEQ ID NO:3) (17 mer "mixed probe") contains as thesingle strand DNA fragment, the sequence information of the BrCN peptideI amino acid position 10-15 (complementary strand). ##STR3##

Oligonucleotide II (SEQ ID NO:4) (42 mer) contains as a single strandDNA fragment, the sequence information of the BrCN peptide I amino acidposition 8-21 (complementary strand). The codon selection was carriedout under the assumption of a guanine(G) and cytosine(C) rich DNAsequence (this takes into consideration guanine(G) and cytosine(C)nucleotides before adenine(A) and thiamine(T) nucleotides on the thirdposition of the triplets). ##STR4##

Oligonucleotide III (SEQ ID NO:5) contains as the single strand DNAfragment sequence, information of the BrCN peptide II (complementarystrand). ##STR5##

By using these oligonucleotides it was possible to localise thecarbamate hydrolase gene within the plasmid pHP52 by hybridization. Forthis, the plasmid DNA was cleaved with restriction endonucleases and theresulting fragments were separated by agarose gel electrophoresis andthen transferred according to the method of E. M. Southern (J. Mol.Biol. 98. 503-517 (1975)) in single strand form on membrane filters(Gene Screen Plus™ hybridising membrane, Du Pont de Nemours/NEN ResearchProducts).

The oligonucleotides were end marked by use of T4-polynucleotide kinase(Boehringer Mannheim) and [γ-³² P]-adenosine-5'-triphosphate (>5000Ci/mmol, Du Pont de Nemours/NEN Research Products) using the method ofR. B. Wallace and C. G. Miyada, Methods in Enzymology 152, 432-442(1987) and treated without further purificiation for the hybridisation.

The hybridization was carried out using standard processes (P. J. Mason& J. G. Williams in "Nucleic Acid Hybridisation" p. 113-160 (1985) B. D.Hames & S. J. Higgins Hrsg. IRL Press Oxford, Washington D.C.). Underthe conditions 6×SSC, 10×Denhardt, 0.5% w/v SDS and 100 u/ml t RNA(B±ckerhefe, Boehringer Mannheim), as well as 10 ng/ml markedoligonucleotides I/II/III at 41° C. (=6 hours), a specific hybridisationcan be achieved. The detection of the hybrids was carried out byautoradiography (T. Maniatis, E. F. Fritsch & J. Sambrook. "MolecularCloning", Cold Spring Harbor Laboratory (1982)).

EXAMPLE 5

Isolation and characterisation of the plasmid pHP52 from Arthrobacteroxidans P52.

For isolation of plasmid pH52 from Arthrobacter, the alkali extractionmethod of Birnboim and Doly (Birnboim H. C. & Doly J. (1979) Nucl AcidRes., 7, 1513-1523) was used, with a modification by Brandsch and Decker(Brandsch, R. & Decker, K. (1984) Arch. Microbiol. 138, 15-17). Forplasmid preparation, the bacteria were cultivated in 6 liters ofLB-medium comprising:

    ______________________________________                                        Bacto-trypton (Difco.sup.R)                                                                        10 g/l                                                   Bacto-Yeast-Extract (Difco.sup.R)                                                                   5 g/l                                                   NaCl                 10 g/l                                                   ______________________________________                                    

to a cell density of OD₅₅₀ =1.4 and harvested by centrifuging.

The cells were resuspended in a total of 210 ml solution I (50 mMglucose: 10 mM EDTA; 25 mM Tris/HCl, pH 8.0; 1 mg/ml lysosyme) andincubated for 1 hour at room temperature. Lysis was carried out byaddition of 360 ml solution II (0.2M NaOH; 1% SDS). After gentle butthorough mixing and subsequent incubation for 5 minutes at roomtemperature, followed by cooling on ice for 5 minutes, the mixture wasneutralised by the addition of 180 ml solution III (2M Tris/HCl, pH7.0/0.5M KCl. After incubation for 1 hour on ice, the undissolvedprecipitate was separated by centrifugation. The plasmid DNA wasprecipitated from the clear supernatant by addition of 0.6 volumesisopropanol and, after an incubation of 15 minutes at room temperature,pelleted by centrifugation (15,000×g/30 minutes). The plasmid-containingprecipitate was dried in vacuo and dissolved in 24 ml 10×TE buffer (100mM Tris/HCl, pH 8.0:10 mM EDTA). This plasmid containing solution wasthen purified by isopycnic cesium chloride density gradient,centrifuging in the presence of Ethidium bromide (Maniatis T., FritschE. F. & Sambrook J. in "Molecular Cloning" (1982), Cold Spring HarborN.Y.).

Purified plasmid DNA was mapped by restriction analysis which cut theplasmid once or gave several fragments. These fragments were resolved byagarose gel electrophoresis (0.8% w/v). Molecular weight standards usedin mapping plasmid DNA were Hind III or Hind III and EcoRI digestedbacteriophage DNA.

The restriction analysis data were consistent with a circular map ofpHP52 (FIG. 4). The size of the plasmid is the sum of individualrestriction fragments.

All the processes were carried out in this Example according to standardmethods (cf. Maniatis T., Fritsch E. F. & Sambrook J. in "MolecularCloning" Cold Spring Harbor. N.Y. (1982)).

EXAMPLE 6

Identification of the coding region of the carbamate hydrolase gene byoligonucleotide hybridisation.

By hybridisation of the restriction fragments Of the plasmid pHP52separated by gel electrophoresis and transferred on membrane filterswith the ³² P marked oligonucleotide described in Example 4, theposition of the coding region of the carbamate hydrolase gene can bedefinitely correlated on the restriction map of the plasmid pHP52. InFIG. 5, the hybridizing area is enlarged. All three oligonucleotideshybridize with the central part of a PstI restriction fragment of size3.3 kb. In FIG. 6, a detailed restriction map of the fragment is shownfrom which the exact positions of the hybridising areas can be seen.

EXAMPLE 7

Cloning of the carbamate hydrolase gene in E. coli and demonstration ofthe genes' expression under lac promoter control.

For cloning the carbamate hydrolase gene in E. coli the vector pUC19(Yanish-Perron, C. Vieira, J. & Messing, J. (1985) Gene 33, 103ff) wasused. The pUC19 DNA was linearised by cleavage with restriction nucleasePstI and treated with alkaline phosphatase. The DNA of the 3.3 kb longPstI restriction fragment of the plasmid pHP52 was isolated (afterdigesting the wild-type plasmid DNA with PstI) by preparative agarosegel electrophoresis. The linearised and dephosphorylated vector DNA andthe 3.3 kb long PstI fragment was then ligated with T4 DNA ligase. E.coli DH5α was transformed with the ligation mixture,

Two types of clones were obtained which contained the fragment indifferent orientations to the transcription direction of the lac Z' geneof the vector pUC 19. These are the clones pp52 Pst and pp52 Pst inv.The restriction map of both clones is shown in FIG. 5. The clones oftype E. coli pp52 Pst express carbamate hydrolase, after addition of theinductor isopropyl-β-D-thiogalactopyranosid to the culture medium.Without inducer addition (repressed state of the lac promotor) tologarithmic cultures of the clone pp52 Pst as well as by repressed andinduced logarithmic cultures of the clone pp52 Pst inv in enzymeextracts, no enzyme activity was seen using the assays described inExample 3.

This means that the carbamate hydrolase gene in clones of type pp52 Pstlies in the same transcription direction (5'-3' orientation) as the lacZ' gene of the vector. The Arthrobacter promoter is not or only slightlyexpressed in E. coli.

EXAMPLE 8

Nucleotide sequence of the carbamate hydrolase gene from theArthrobacter oxidans (species P52) and the deduced protein sequence.

The nucleotide sequence of the carbamate hydrolase gene was establishedby the method of Sanger (Sanger F., Nicklen S. & Coulson A. (1977) Proc.Natl. Acad. Sci. USA. 74. 5463-5468).

For this, 15 sub-clones in the single stranded DNA bacteriophage M13mp18 and M13 mp19 from the pp52 PST DNA were constructed (Messing, J.(1983) Methods in Enzymol. 101, 20-78). After transfection of E coliDH5αF', the sequence of the single stranded recombinant DNA wasestablished.

In FIG. 6, the sequencing strategy of the carbamate hydrolase gene isshown. Altogether the sequence of 1864 base pairs was established.

In FIG. 7(SEQ ID NO:6), the established nucelotide sequence is shownwith the deduced amino acid sequence of the carbamate hydrolase. Thereading frame is clearly defined as described by the amino acidsequences of two BrCN cleavage peptides as described in Example 4. Thereading frame finishes with a TGA stop codon (nucleotide position1789-1791 in FIG. 7 (SEQ ID NO:6)). As a translation start codon, a GTG(nucleotide position 310-312) is suitable. This gives the longest openreading frame of 1479 bp (=493 amino acids). All open reading frameswhich begin with the usual ATG start codons give no protein of suitablesize (compared to the molecular weight determination of the protein).

The hypothesis that translation starts from GTG (position 310-312) isfurther supported by the existence of a definite homologous region tothe consensus sequence for ribosomal E. coli binding sites 7 bp upstreamof the putative GTG start codon.

All cloning steps were carried out by standard processes (cf Maniatis,T., Fritsch, E. F. & Sambrook, J. (1982) in "Molecular Cloning", ColdSpring Harbor, N.Y.). The sequencing reactions were carried out usingSequenase® DNA Sequencing Kits (United States Biochemical Corporation)according to information by the producer. The separation of the markedreaction products was carried in 6% w/v polyacrylamide/urea gel (Maxam,A. H. & Gilbert, W. (1980) Methods Enzymol. 65, 497-559).

EXAMPLE 9

Construction of plasmids for the expression of carbamate hydrolase inplants.

a) Construction of intermediate vectors

Plasmid DNA from pp52Pst (FIG. 8) is methylated using TaqI Methylase (M.TaqI) to make the single SaII restriction site inaccessible to HincII.Then the methylated plasmid is cut with both restriction enzymes XbaIand HincII to release a 2.2 kb DNA fragment containing the open readingframe for carbamate hydrolase. This fragment is purified by preparativeagarose gel electrophoresis. The vector plasmid pA5 (FIG. 8) is cut withXbaI and HincII and then ligated with the purified 2.2 kb DNA fragment,thus linking the 3'-end of the carbamate hydrolase coding region to thepolyadenylation signal of the octopine synthetase (OCS) gene (Dhaese etal., EMBO J 2:419, 1983). Competent cells of E. coIi DH5alpha aretransformed with the recombined DNA and clones are selected on LB-agarcontaining 100 μg/ml carbenicillin. Clones containing the recombinedplasmid pUPA1014 (FIG. 8) are identified by restriction analysis ofisolated plasmid DNA.

Two oligonucleotides of the following sequence are synthesized by anautomatic synthesizer:

1.) 5'-CTAGAGATCT CAACAATGGT TACCAGACCG ATCGCCCACA CCACCGCTGG G-3'(SEQID NO:8)

2.) 5'-GTCCCCAGCG GTGGTGTGGG CGATCGGTCT GGTAACCATT GTTGAGATC-3'(SEQ IDNO:9)

They represent complementary DNA strands which are able to reconstitutethe N-terminal portion of the open reading frame of the carbamatehydrolase gene upstream (5') of the PpuMI restriction site.

Both complementary oligonucleotides are mixed, phosphorylated bypolynucleotide kinase and then annealed by shifting temperature from 70°C. to room temperature overnight. Plasmid DNA of pUPA1014 is digestedwith XbaI and then ligated with the annealed oligonucleotide. Thiscovalently links two olig nucleotides at their XbaI-compatible ends toboth ends of the linearised plasmid. The linear ligation product ispurified by preparative agarose gel electrophoresis and digested withPpuMI to remove the N-terminal part of the coding region. The linear DNAis then recircularized by ligase treatment. Competent cells of E. coliDH5alpha are transformed with the recombined DNA and clones are selectedon LB-agar containing 50 μg/ml carbenicillin. Clones containing therecombined plasmid pUP01015 (FIG. 9) are identified by restrictionanalysis of isolated plasmid DNA. The correct ligation of theoligonucleotide is verified by sequence analysis of purified pUP01015DNA using the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. USA74:5463, 1977) modified for plasmid DNA as template (Chen and Seeburg,DNA 4:165, 1985).

b) Construction of plasmids for the cytoplasmatic expression ofcarbamate hydrolase.

Plasmid DNA of pUP01015 is digested with both restriction enzymes XbaIand HindIII to create a DNA fragment that contains the whole recombinantcoding region of the carbamate hydrolase together with thepolyadenylation signal described above. The fragment is then ligatedwith the plant expression vector plasmid pA8 (A. v. Schaewen,Dissertation, FU Berlin, 1989; FIG. 10) which was similarily treatedwith XbaI and HindIII. The ligation links the gene in correctorientation to the cauliflower mosaic virus (CaMV) 35S promoter(Paszkowski et al., EMBO J. 3:2717, 1984) contained in pA8. Competentcells of E. coli DH5alpha are transformed with the recombined DNA andclones are selected on LB-agar containing 25 μg/ml streptomycin. Clonescontaining the recombined plasmid pMCP01021 (FIG. 10) are identified byrestriction analysis of isolated plasmid DNA.

Plasmid DNA of pMCP01021 is digested with both restriction enzymes EcoRIand HindIII to create a DNA fragment that contains the CaMV 35Spromoter, the carbamate hydrolase coding region and the OCSpolyadenylation signal. The fragment is then ligated with a EcoRI andHindIII cleaved vector plasmid pBIN19 that is part of the binarytransformation system described by Bevan, Nucl. Acids Res. 12:8711,1984. Competent cells of E. coli S17-1 (Simon et al. Bio/Technology1:784-791, 1983) are transformed with the recombined DNA and clones areselected on LB-agar containing 50 μg/ml kanamycin. Clones containing therecombined plasmid pBCP01027 are identified by restriction analysis ofisolated plasmid DNA.

c) Construction of plasmids for the extracellular expression ofcarbamate hydrolase.

Plasmid pA22 (FIG. 11) contains a synthetic intronless sequence thatcodes for the signal peptide of the proteinase inhibitor II(PI) frompotato and can be attached to the N-terminus of other genes to directthe gene product into the endoplasmatic reticulum of the plant cell. Tocreate an intermediate vector for targeting of carbamate hydrolase, theDNA fragment encoding the signal peptide is excised from plasmid pA22 bycleavage with restriction enzymes KpnI and XbaI and then ligated with aKpnI and XbaI cleaved plasmid pA8. Competent cells of E. coli DH5alphaare transformed with the recombined DNA and clones are selected onLB-agar containing 25 μg/ml streptomycin. Streptomycin resistant clonesare then screened for the loss of plasmid pA22 on LB-agar containing 50μg/ml carbenicillin. Carbenicillin sensitive clones are analyzed byrestriction analysis of isolated plasmid DNA and recombined plasmidscontaining the signal peptide encoding sequence are designated pmA 1017(FIG. 11)

Plasmid DNA of pUP01015 is digested with both restriction enzymesHindIII and BgIII to create a DNA fragment that contains the wholerecombinant coding region of the carbamate hydrolase together with thepolyadenylation signal described above. The fragment is then ligatedwith the plant expression vector plasmid pMA1017 which has been digestedwith BamHI and HindIII. The ligation links the gene in correctorientation to the cauliflower mosaic virus (CaMV) 35S promotor(Paszkowski et al., EMBO 3.3:2717, 1984) and the proteinase inhibitorsignal sequence described above. Competent cells of E. coli DH5alpha aretransformed with the recombined DNA and clones are selected on LB-agarcontaining 25 μg/ml streptomycin. Clones containing the recombinedplasmid pMAP01022 (FIG. 12) are identified by restriction analysis ofisolated plasmid DNA.

Plasmid DNA of pMAP01022 is digested with both restriction enzymes EcoRIand HindIII to create a DNA fragment that contains the CaMV 35Spromoter, the PI signal sequence linked in frame to the carbamatehydrolase coding region and the OCS polyadenylation signal. The fragmentis then ligated with a EcoRI and HindIII cleaved vector plasmid pBIN19(Bevan, Nucl. Acids Res. 12:8711, 1984). Competent cells of E. coliS17-1 (Simon et al. Bio/Technology 1:784-791, 1983) are transformed withthe recombined DNA and clones are selected on LB-agar containing 50μg/ml kanamycin. Clones containing the recombined plasmid pBAP01027 areidentified by restriction analysis of isolated plasmid DNA.

EXAMPLE 10

Transformation of tobacco with chimeric carbamate hydrolase genes

a) Transfer of recombinant carbamate hydrolase from E. coil to A.tumefaciens

Strains of E. coli S17-1 containing chimeric carbamate hydrolase geneson plasmids pBCP01027 or pBAP01028 are grown at 37° C. in liquid LBmedium containing 50 μg/ml kanamycin. Aqrobacterium tumefaciens LBA4404(Bevan, Nucl. Acids Res. 12:8711, 1984) is grown at 28° C. in liquid YEBmedium (Yeast extract 1 g/l, beef extract 5 g/l, peptone 5 g/l, sucrose5 g/l, sucrose 5 g/l, MgSO₄ 0.5 g/l). 0.4 ml samples of E. coli cultureare centrifuged and the bacterial pellets are resuspended in 0.4 ml YEB.Bacterial suspensions of E. coli in YEB and samples of Aqrobacteriumculture in YEB are then mixed in a 1:1 ratio in relation to celldensity. Samples of50-100 μl from the mixtures are spotted onto LB-agarand incubated at 28° C. for 6-16 hours. Bacterial mating mixtures thathave grown on the agar are suspended in liquid M9-salts (6 g/l Na₂ HPO₄,3 g/l KH₂ PO₄, 0.5 g/l NaCl, 1 g/l NH₄ Cl, 2 mM MgSO₄, 0.1 mM CaCl₂, 1mM thiamine. HCl) and then plated in several dilutions onto M9-agarcontaining 2 g/l sucrose and 50 μg/ml kanamycin. Plates are incubated at28 ° C. for several days until bacterial colonies have grown. Thesecolonies are further purified by subsequent cultivation on the samemedium. That these clones of A. tumefaciens LBA4404 contain recombinantplasmids pBCP01027 and pBAP01028 respectively is verified by restrictionanalysis of isolated plasmid DNA.

b) Transfer of recombinant carbamate hydrolase from A. tumefaciens totobacco.

For transformation of tobacco, A. tumefaciens strains harbouringcarbamate hydrolase plasmids are grown overnight at 28° C. in liquid YEBmedium containing 50 μg/ml kanamycin. Cells are centrifuged at 5000 gfor 15 minutes and resuspended in the same volume of YEB withoutantibiotic. Nicotiana tabacum Wisconsin W38 plantlets are grown understerile conditions on solid MS medium containing 20 g/l sucrose. Leavesare cut from those plants, dissected into pieces of around 1 cm² andrinsed with the bacterial suspension. Leaf disks are then placed ontosolid MS medium containing 20 g/l sucrose. After 2 days of incubation atroom temperature in the dark, leaf disks are transferred to solid MSmedium containing 16 g/l glucose, 1 mg/l benzylaminopurine, 0.2 mg/lnaphthylacetic acid, 500 mg/l claforan and 50 mg/l kanamycin. Incubationis continued at 25° C. under a daily regime of 16 hours light(photosynthetically active radiation=67 μEM⁻² S⁻¹ ) and 8 hours dark.The medium is changed every week until shoots appear. These are cut fromthe callus and transferred to MS medium containing 20 g/l sucrose and250 mg/l claforan. Incubation is continued under the same conditionsuntil roots of 1-2 cm in length have formed and plants are transferredto soil. Total RNA isolated from leaves is analyzed by northern blothybridisation using the 1.8 kb EcoRI-HindIII fragment of pUP01015 as alabeled probe. Transformed plants synthesize a transcript of around 1.8kb in size that specifically hybridizes with the carbamate hydrolasecooing sequence.

EXAMPLE 11

Detection of transformed plants which are resistant to the herbicidalactivity of phenmedipham.

Transgenic plants are transferred to soil and grown in a growth chamberat 25° C. with a day/night rhythm of 16 hours light and 8 hours dark. Nodifference in growth can be seen between transformed and untransformedtobacco. Plants that have a leaf length of around 10 cm are sprayed withthe herbicide Betanal® (active ingredient: 157 g/l phenmedipham).

Doses corresponding to field application rates of 1 kg/ha, 3 kg/ha and10 kg/ha are used to distinguish between resistant plants anduntransformed wildtype plants: Whereas 1 kg/ha is completely lethal forwildtype plants, transgenic plants which express the carbamate hydrolasegene show resistance levels between 1 kg/ha and 10 kg/ha (FIG. 13 and14).

The same spraying experiment is done by using Betanal® AM(activeingredient: 157 g/l desmedipham) as the herbicidal agent.

EXAMPLE 12

Analysis of herbicide detoxification in plants sprayed withphenmedipham.

Transgenic tobacco plants expressing carbamate hydrolase anduntransformed control plants are grown as described in example 11 andsprayed with the herbicide Betanal® corresponding to 1 kg/haphenmedipham. Normalized variable fluorescence is measured on intactleaves of sprayed plants as is described by Voss, Weed Science32:675-680 (1984). The equipment used is a Kompakt-Fluorometer RKF 1000(Ingenieurburo F. U. R. Dr. H. Voss, Berlin). Measurement values beforespraying are taken as 100% relative variable fluorescence. Subsequentmeasurements are performed in a time course of 2, 4, 8, 24 hours andthen every day up to 4 days. Relative variable fluorescence values oftransgenic tobacco plants expressing carbamate hydrolase stay constantlyhigher than 90%, in contrast values from untransformed tobacco fallbelow 10% within the first 8 hours after spraying (FIG. 15).

The same spraying experiment is done by using Betanal® AH (activeingredient: 157 g/l desmedipham) as the herbicidal agent.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Arthrobacter oxidans                                            (B) STRAIN: P52                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerAspGluPheAlaAsnLeuAspArgTrpThrGlyLysProPheVal                              1510 15                                                                       XaaXaaLeuAspGluValAlaVal                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                          (v) FRAGMENT TYPE: internal                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Arthrobacter oxidans                                            (B) STRAIN: P52                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GluHisThrLysXaaXaaGluXaaProLeuAlaPheTyrProValPhe                              15 1015                                                                       AsnGlu                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Arthrobacter oxidans                                           (B) STRAIN: P52                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AANGGYTTNCCNGTCCA17                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Arthrobacter oxidans                                            (B) STRAIN: P52                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CTGGTCCAGGCCGTCCACGAACGGCTTGCCGGTCCAGCGGTC42                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: YES                                                       (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Arthrobacter oxidans                                            (B) STRAIN: P52                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TTCGTT GAAGACCGGGTAGAACGC24                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1864 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv ) ANTI-SENSE: NO                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: RBS                                                             (B) LOCATION: 298..302                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 310..1791                                                       (D) OTHER INFORMATION: /codon=(seq: "gtg", aa: Val)                           /product="carbamate hydrolase"                                                /translexcept=(pos: 310 .. 312, aa: Met)                                      /note="terminator (1789-1791)"                                                (ix ) FEATURE:                                                                (A) NAME/KEY: matpeptide                                                      (B) LOCATION: 310..1788                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TCCTTGCCAGTCACGGCACCCCAGCCAACCCGGAAGTGGCACCTGCTCGGGCACATCGGT60                GCGAACGCTTCGTCCTGATTCCGATGCCAACTGCTTGACGGCCGTGACACATATGTAGCA1 20              TAGTCGCCTAGCATGGACCCGCAGCACACCTGCTGTCGGCTCCCGCGCTATCCCCGACCA180               GCGCCGGTCACGGGTAGTCCTCGTGAGAGGCACCAGAACGACAACGGCGCACTGTCCCGC240               AACACGGCCGTATAACCCCACCGGGGTCCGCGCCCGAGCTA GTTCTGGCTCAACCATAAG300              GAGAACCTCGTGATTACCAGACCGATCGCCCACACCACCGCTGGGGAC348                           MetIleThrArgProIleAlaHisThrThrAlaGlyAsp                                       15 10                                                                         CTCGGCGGTTGCCTTGAAGACGGCCTGTACGTGTTCCGAGGAGTGCCG396                           LeuGlyGlyCysLeuGluAspGlyLeuTyrValPheArgGlyValPro                              1520 25                                                                       TACGCCGAGCCGCCGGTCGGCGACCTGCGGTGGCGGGCGGCGCGCCCG444                           TyrAlaGluProProValGlyAspLeuArgTrpArgAlaAlaArgPro                              303540 45                                                                     CACGCCGGCTGGACCGGCGTCCGCGACGCCTCCGCGTATGGTCCCTCG492                           HisAlaGlyTrpThrGlyValArgAspAlaSerAlaTyrGlyProSer                              5055 60                                                                       GCGCCGCAACCCGTGGAGCCTGGCGGCTCGCCGATCCTTGGGACACAC540                           AlaProGlnProValGluProGlyGlySerProIleLeuGlyThrHis                              6570 75                                                                       GGCGACCCTCCGTTTGACGAGGACTGCCTGACTCTCAATCTTTGGACC588                           GlyAspProProPheAspGluAspCysLeuThrLeuAsnLeuTrpThr                              8085 90                                                                       CCGAACCTCGACGGCGGTAGCCGGCCGGTCCTCGTCTGGATCCATGGT636                           ProAsnLeuAspGlyGlySerArgProValLeuValTrpIleHisGly                              95100105                                                                      GGGGG CCTACTAACCGGCTCGGGAAATCTACCTAACTACGCGACCGAT684                          GlyGlyLeuLeuThrGlySerGlyAsnLeuProAsnTyrAlaThrAsp                              110115120125                                                                  A CCTTCGCCCGCGACGGCGACTTGGTAGGTATCTCAATCAATTACCGG732                          ThrPheAlaArgAspGlyAspLeuValGlyIleSerIleAsnTyrArg                              130135140                                                                      CTCGGGCCTCTTGGATTCCTCGCAGGAATGGGCGACGAGAATGTCTGG780                          LeuGlyProLeuGlyPheLeuAlaGlyMetGlyAspGluAsnValTrp                              145150155                                                                     CTC ACCGATCAGGTAGAGGCACTGCGCTGGATTGCAGATAACGTTGCT828                          LeuThrAspGlnValGluAlaLeuArgTrpIleAlaAspAsnValAla                              160165170                                                                     GCCTTCGG TGGAGACCCGAACCGGATCACTCTCGTCGGTCAATCAGGC876                          AlaPheGlyGlyAspProAsnArgIleThrLeuValGlyGlnSerGly                              175180185                                                                     GGGGCATACTCGATCG CAGCGCTCGCCCAACACCCGGTCGCCCGTCAG924                          GlyAlaTyrSerIleAlaAlaLeuAlaGlnHisProValAlaArgGln                              190195200205                                                                  CTGTTCCACCGC GCGATCCTACAAAGCCCACCATTCGGGATGCAACCC972                          LeuPheHisArgAlaIleLeuGlnSerProProPheGlyMetGlnPro                              210215220                                                                     CATACAGTTGAA GAATCGACGGCAAGGACGAAGGCCCTGGCCCGGCAT1020                         HisThrValGluGluSerThrAlaArgThrLysAlaLeuAlaArgHis                              225230235                                                                     CTCGGGCACGATGA CATCGAGGCCCTGCGCCATGAGCCGTGGGAGAGG1068                         LeuGlyHisAspAspIleGluAlaLeuArgHisGluProTrpGluArg                              240245250                                                                     CTGATTCAAGGCACGATAG GCGTCCTGATGGAACACACCAAATTTGGC1116                         LeuIleGlnGlyThrIleGlyValLeuMetGluHisThrLysPheGly                              255260265                                                                     GAATGGCCCCTGGCATTCTATCCGGTG TTCGATGAGGCAACGATACCT1164                         GluTrpProLeuAlaPheTyrProValPheAspGluAlaThrIlePro                              270275280285                                                                  CGCCATCCGATTGAGTCCATTATC GATTCCGACATCGAAATCATCATC1212                         ArgHisProIleGluSerIleIleAspSerAspIleGluIleIleIle                              290295300                                                                     GGCTGGACACGCGACGAGGGCAC TTTTCCGTTTGCCTTCGACCCTCAG1260                         GlyTrpThrArgAspGluGlyThrPheProPheAlaPheAspProGln                              305310315                                                                     GTTTCACAGGCGGATCGCGATCAGG TCGAGTCATGGTTGCAGAAGCGT1308                         ValSerGlnAlaAspArgAspGlnValGluSerTrpLeuGlnLysArg                              320325330                                                                     TTCGGAGACCACGCCGCCTCGGCCTACGAG GCTCACGCCGGCGACGGA1356                         PheGlyAspHisAlaAlaSerAlaTyrGluAlaHisAlaGlyAspGly                              335340345                                                                     ACCAGTCCTTGGACCGTTATCGCCAACGTTGTGGGCGAC GAGCTCTTT1404                         ThrSerProTrpThrValIleAlaAsnValValGlyAspGluLeuPhe                              350355360365                                                                  CACAGCGCTGGGTACCGGGTCGCGGACGAACGGGC AACGCGCAGACCG1452                         HisSerAlaGlyTyrArgValAlaAspGluArgAlaThrArgArgPro                              370375380                                                                     GTACGGGCCTATCAGTTCGACGTAGTCTCGCCCT TGTCGGACGGAGCC1500                         ValArgAlaTyrGlnPheAspValValSerProLeuSerAspGlyAla                              385390395                                                                     CTCGGCGCGGTCCACTGCATCGAAATGCCGTTCACA TTTGCCAATCTC1548                         LeuGlyAlaValHisCysIleGluMetProPheThrPheAlaAsnLeu                              400405410                                                                     GACCGTTGGACGGGGAAGCCGTTCGTGGACGGCCTGGATCCA GACGTG1596                         AspArgTrpThrGlyLysProPheValAspGlyLeuAspProAspVal                              415420425                                                                     GTGGCTCGGGTGACCAACGTGTTGCATCAGGCCTGGATCGCATTCGTC 1644                         ValAlaArgValThrAsnValLeuHisGlnAlaTrpIleAlaPheVal                              430435440445                                                                  CGAACGGGAGACCCCACGCACGACCAGTTGCCGGTGTGGCCAACGT TC1692                         ArgThrGlyAspProThrHisAspGlnLeuProValTrpProThrPhe                              450455460                                                                     CGAGCGGACGACCCAGCGGTGTTGGTCGTCGGCGACGAGGGAGCA GAG1740                         ArgAlaAspAspProAlaValLeuValValGlyAspGluGlyAlaGlu                              465470475                                                                     GTGGCGCGGGATCTAGCGCGCCCGGACCACGTCAGCGTTCGGACCCTA 1788                         ValAlaArgAspLeuAlaArgProAspHisValSerValArgThrLeu                              480485490                                                                     TGAGGGTCGCGGGTCGCCGGGGTCTTGAGGCCGGAGGGCCTCGCGTATGCAGTGATTCGT18 48             GGATCACCGGCCAGTT1864                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 493 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetIleThr ArgProIleAlaHisThrThrAlaGlyAspLeuGlyGly                             151015                                                                        CysLeuGluAspGlyLeuTyrValPheArgGlyValProTyrAlaGlu                              20 2530                                                                       ProProValGlyAspLeuArgTrpArgAlaAlaArgProHisAlaGly                              354045                                                                        TrpThrGlyValArgAspAlaSerAlaTyrGl yProSerAlaProGln                             505560                                                                        ProValGluProGlyGlySerProIleLeuGlyThrHisGlyAspPro                              6570758 0                                                                     ProPheAspGluAspCysLeuThrLeuAsnLeuTrpThrProAsnLeu                              859095                                                                        AspGlyGlySerArgProValLeuValTrpIleHisGlyGlyGlyLeu                               100105110                                                                    LeuThrGlySerGlyAsnLeuProAsnTyrAlaThrAspThrPheAla                              115120125                                                                     ArgAspGlyAspLeuValGly IleSerIleAsnTyrArgLeuGlyPro                             130135140                                                                     LeuGlyPheLeuAlaGlyMetGlyAspGluAsnValTrpLeuThrAsp                              145150155 160                                                                 GlnValGluAlaLeuArgTrpIleAlaAspAsnValAlaAlaPheGly                              165170175                                                                     GlyAspProAsnArgIleThrLeuValGlyGlnSerGlyGl yAlaTyr                             180185190                                                                     SerIleAlaAlaLeuAlaGlnHisProValAlaArgGlnLeuPheHis                              195200205                                                                     ArgAlaIle LeuGlnSerProProPheGlyMetGlnProHisThrVal                             210215220                                                                     GluGluSerThrAlaArgThrLysAlaLeuAlaArgHisLeuGlyHis                              225230 235240                                                                 AspAspIleGluAlaLeuArgHisGluProTrpGluArgLeuIleGln                              245250255                                                                     GlyThrIleGlyValLeuMetGluHisThr LysPheGlyGluTrpPro                             260265270                                                                     LeuAlaPheTyrProValPheAspGluAlaThrIleProArgHisPro                              275280285                                                                     IleGluSerIleIleAspSerAspIleGluIleIleIleGlyTrpThr                              290295300                                                                     ArgAspGluGlyThrPheProPheAlaPheAspProGlnValSerGln                              305 310315320                                                                 AlaAspArgAspGlnValGluSerTrpLeuGlnLysArgPheGlyAsp                              325330335                                                                     HisAlaAlaSerAlaTyr GluAlaHisAlaGlyAspGlyThrSerPro                             340345350                                                                     TrpThrValIleAlaAsnValValGlyAspGluLeuPheHisSerAla                              355360 365                                                                    GlyTyrArgValAlaAspGluArgAlaThrArgArgProValArgAla                              370375380                                                                     TyrGlnPheAspValValSerProLeuSerAspGlyAlaLeuGlyAla                              38 5390395400                                                                 ValHisCysIleGluMetProPheThrPheAlaAsnLeuAspArgTrp                              405410415                                                                     ThrGlyL ysProPheValAspGlyLeuAspProAspValValAlaArg                             420425430                                                                     ValThrAsnValLeuHisGlnAlaTrpIleAlaPheValArgThrGly                              435 440445                                                                    AspProThrHisAspGlnLeuProValTrpProThrPheArgAlaAsp                              450455460                                                                     AspProAlaValLeuValValGlyAspGluGlyAlaGlu ValAlaArg                             465470475480                                                                  AspLeuAlaArgProAspHisValSerValArgThrLeu                                       485490                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 51 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Arthrobacter oxidans                                            (B) STRAIN: P52                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CTAGAGATCTCAACAATGGTTAC CAGACCGATCGCCCACACCACCGCTGGG51                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GTCCCCAGCGGTGGTGTGGGCGATCGGTCTGGTAACCATTGTTGAGATC49                       

We claim:
 1. A chimeric carbamate hydrolase gene comprising regulatoryregions which are functional in plant cells, operably linked to acarbamate hydrolase gene having the activity of a carbamate hydrolasefrom Arthrobacter oxidans.
 2. A chimeric carbamate hydrolase geneaccording to claim 1, wherein the carbamate hydrolase gene comprises thesequence of FIG. 7, identified as SEQ ID NO:
 6. 3. A plant that containsthe chimeric carbamate hydrolase gene according to claim 2,characterized in that the plant expresses carbamate hydrolase activity.4. A plant according to claim 3, wherein the plant is tobacco.
 5. Thechimeric carbamate hydrolase gene according to claim 1 , wherein theArthrobacter oxidans is P 16/4/B (DSM 4038).
 6. The chimeric carbamatehydrolase gene according to claim 1, wherein the Arthrobacter oxidans isP 67 (DSM 4039).
 7. The chimeric carbamate hydrolase gene according toclaim 1, wherein the Arthrobacter oxidans is P 75 (DSM 4040).
 8. Thechimeric carbamate hydrolase gene according to claim 1, wherein theArthrobacter oxidans is P 11/1/-b (DSM 4041).
 9. The chimeric carbamatehydrolase gene according to claim 1, wherein the Arthrobacter oxidans isP 15/4/A (DSM 4045).
 10. The chimeric carbamate hydrolase gene accordingto claim 1, wherein the Arthrobacter oxidans is P 21/2 (DSM 4046). 11.The chimeric carbamate hydrolase gene according to claim 1, wherein thecarbamate hydrolase shows a pH optimum of 6.8, a molecular weight in therange of 50-60 kd and an isoelectric point of pI=6.2.
 12. A method oftransforming a plant comprisingtransforming plant cells with a chimericcarbamate hydrolase gene to produce transformants, wherein the chimericcarbamate hydrolase gene comprises regulatory regions which arefunctional in plant cells, operably linked to a carbamate hydrolase genehaving the activity of a carbamate hydrolase gene from Arthrobacteroxidans; selecting for transformants expressing the chimeric carbamatehydrolase gene; and growing the transformants expressing the chimericcarbamate hydrolase gene to produce plants.